Cross-linked polysaccharide has in different forms become very important as a gel matrix for chromatographic separation, especially for biomolecules such as proteins, nucleic acids etc.
The chromatographic separation is carried out in a column packed with the matrix through which a liquid flow is forced. A sample which can be a mixture of different proteins, is introduced at the top of the column and then moves with the flow through the column. The proteins will be retarded on the matrix in such a manner that proteins having different properties, e.g., size, will be retarded differently and therefore separated.
By varying the concentration of polysaccharide when producing these gel matrices, different pore sizes can be obtained, the lower the concentration, the bigger pores, i.e., the higher exclusion limit. The size of the pores in proportion to the size of the biomolecules to be separated, is decisive for the separation.
The technique most known for the separation of biomolecules is based on diffusion of molecules into and out of these pores. At a fixed pore size, molecules that are bigger than the pores will be completely excluded, and therefore quickly elute from the column. If the protein mixture contains molecules which are smaller than these pores, a diffusion of the molecules into the pores will take place, these molecules being retarded. Due to differences of size of the molecules which diffuse into the pores, a greater or lesser retardation is obtained, the result being a separation according to the size of the molecules. This type of separation is called molecular sieving or gel filtration. However, it is assumed that the part of the gel matrix which is exposed to the molecules does not have any other interacting properties, i.e., the gel matrix shall be completely inert to the molecule mixture which is being chromatographed.
In other separation techniques, such as ion exchange, hydrophobic interaction, affinity etc., either properties that are naturally in the gel matrix or properties which, through a chemical change of the gel matrix, have been introduced into the pores, are utilized.
The separation technique has in recent years developed towards shorter separation times and higher resolution, this has led to rigid, small (5-10 micrometers) particles being used as matrices. Due to their softness, traditional polysaccharides have been produced in particle sizes from 40 micrometers upwards, and in spite of this, the separation has in some cases taken 10-20 hours to carry out. This is especially true of gel filtration and in particular when the exclusion limit has been high. This limit depends on the concentration of the polysaccharide as mentioned earlier.
Another common matrix which fulfills these new requirements is silica based gels. Silica is however not stable in aqueous solutions especially at a higher pH value (greater than 7.5). Since most separations of biomolecules are carried out in aqueous solution, this is unsatisfactory. Moreover, silica contains silanol groups which themselves have an ion exchange and/or strongly hydrogen bonding effect.
The traditional polysaccharide gel matrices can be stabilized by chemically cross-linking the polymer chains with each other. This cross-linking takes place between hydroxyl groups available on the polysaccharides and has been utilized for example in connection with agarose which has been cross-linked with epichlorhydrin (U.S. Pat. No. 3,507,851) and other bifunctional reagents (U.S. Pat. No. 3,860,573). In this context, a bifunctional reagent is a chemical compound which under the same conditions can react with both its functional groups. Other bifunctional reagents used are bis-epoxides, divinyl sulphon and dicarboxylic acid chlorides (GB 1352613).
Agar and agarose and some similar polysaccharides will form gels at fixed temperatures. Upon gelification, a macroporous gel is obtained and the size of the pores is determined as mentioned earlier by the concentration of polysaccharide. The polysaccharide chains will then by hydrogen bonding, attain certain fixed distances and form chain-like structures between which the pores are formed. By cross-linking within these structures, the size of the pores is not affected, only the rigidity of the particle J. Porath et al (J of Chromatography 103 (1975) 49-62) has studied the influence of cross-linking agents having different chain lengths on the properties of the agarose gels and found that a gel matrix of a considerably improved rigidity was obtained with cross-linking agents where the inserted molecule chain has a length of 5 atoms. A crosslinking agent which fulfilled these requirements was divinylsulphone which is a homobifunctional cross-linking agent.
In the European patent application No. 84850215.9 the use of homopolyfunctional cross-linking agents is described and it is shown that a further extension increase in the number of cross-linking atoms improves the rigidity
The disadvantages of using such homobifunctional and homopolyfunctional cross-linking agents are several. One essential requirement is that the cross-linking agent shall be as hydrophilic as possible, because with increasing distance between the hydrophilic functional groups the hydrophilicity is reduced. This can be compensated for by using cross-linking agents containing hydroxyl groups or those which after cross-linking form hydroxyl groups. However, these hydroxyl groups affect the cross-linking. As mentioned earlier the cross-linking takes place between hydroxyl groups available on the polysaccharides. It has been shown by L Holmberg (Doctorial Thesis, Swedish University of Agricultural Sciences 1983, pp. 28-29) that upon cross-linking of polysaccharides with epichlorhydrin very little monomer cross-linking takes place, i.e., a crosslinking where only monomer epichlorhydrin participates. On the contrary, Holmberg points out that the main part (&gt;98%) is polymer cross-linking and/or substitution. Thus, it is seen that during the course of the cross-linking reaction one does not know the number of the atoms in the cross-linking bridge obtained. This is also shown by the result with divinylsulphon (J Porath, J of Chromatogr 103 (1975) 49-62), which shows low reproducibility as regards pressure/flow.
The homopolyfunctional cross-linking agents are even more hydrophobic than epichlorhydrin. Upon cross-linking, epichlorhydrin gives rise to the most hydrophilic bridge and should therefore be ideal. However, polymer cross-linking is obtained.